Virulence determinants are factors which play a major pathogenic roles in many infections. While there is very little information available on the existence of virulence factors in the periodontopathic organisms disease they almost certainly do exist. The present study concerns one of these organisms, Actinobacillus actinomycetemcomitans. Detailed pathogenic mechanisms of A. actinomycetemcomitans infections are not known, but the organism produces a variety of potential virulence factors including a leukotoxin capable of killing human polymorphonuclear leukocytes and macrophages in vitro. If the toxin has similar effects on PMNs and MNs in the human gingival crevice it could represent a "strategy" for A. actinomycetemcomitans survival and virulence because it could impair both innate and adaptive host defense systems. We have isolated and purified the leukotoxin from A. actinomycetemcomitans and have cloned the toxin genes into E. coli. The primary objective of the present application is to utilize molecular biology techniques to relate the structure of the leukotoxin to its function. The project has four major areas of investigation: 1) To determine the structure and transcriptional organization of the genes in the leukotoxin gene complex; 2) To study the structure/function relationships of the leukotoxin A gene product; 3) To determine the functions of the Leukotoxin B, C, and D Gene Products; and 4) To compare the organization, expression and function of the genes of the leukotoxin gene cluster in leukotoxic and nonleukotoxic strains of A. actinomycetemcomitans. Identifying and understanding the role of virulence factors in periodontal disease will represent a major advance in our understanding of mechanisms of tissue destruction in this disease. As a sidelight, the proposed studies may also provide information on the basic biology of the macrophage/granulocyte. In this regard, an intriguing problem is the reason for the high cell selectivity exhibited by the A. actinomycetemcomitans toxin which appears to be achieved in the absence of a classic receptor mediated mechanism. Whatever mechanisms are involved in its action, the A. actinomycetemcomitans leukotoxin may be a therapeutically useful agent, because of its specificity We have shown that it is capable of killing leukemia cells related to promyelocytes, and it may be possible to use the recombinantly synthesized product in the treatment of some leukemias. In a more general way, elucidation of the precise mechanisms involved in cell specificity and killing may lead to a new class o therapeutic agent. These may be protein in nature or organic compounds based upon information gained from study these intriguing systems.